Dendritic cells play no significant role in the laser-induced choroidal neovascularization model

Age-related macular degeneration (AMD) is genetically associated with complement. Dendritic cells (DCs) play key roles during innate and adaptive immunity, and express complement components and their receptors. We investigated ocular DC heterogeneity and the role of DCs in the laser-induced choroidal neovascularization (CNV) model. In order to determine the function of DCs, we used two models of DC deficiency: the Flt3−/− and Flt3l−/− mouse. We identified three types of ocular DCs: plasmacytoid DC, classical DC-1, and classical DC-2. At steady-state, classical DCs were found in the iris and choroid but were not detectable in the retina. Plasmacytoid DCs existed at very low levels in iris, choroid, and retina. After laser injury, the number of each DC subset was up-regulated in the choroid and retina. In Flt3−/− mice, we found reduced numbers of classical DCs at steady-state, but each DC subset equally increased after laser injury between wildtype and Flt3−/− mice. In Flt3l−/− mice, each DC subsets was severely reduced after laser injury. Neither Flt3−/− or Flt3l−/− mice demonstrated reduced CNV area compared to wildtype mice. DCs do not play any significant role during the laser-induced CNV model of neovascular AMD.


Results
Multi-parameter flow cytometry of ocular DCs. We used multi-parameter flow cytometry to analyze dendritic cell heterogeneity in murine eyes at steady state and after laser-induced CNV. Whole mouse eyes were dissected to remove conjunctiva, orbital tissue, extraocular muscles, and optic nerve. Cornea, sclera, iris, ciliary body, lens, vitreous, retina, and choroid were chemically and mechanically digested for multi-parameter flow cytometry at steady-state or 3 days post laser injury. CD45 + cells were identified from singlet, live cells and gated forward (Fig. 1A, H). T-cells (CD4 and CD8), B-cells (B220), NK cells (NK1.1), eosinophils (SiglecF), and neutrophils (Ly6G) were excluded using a lineage (Lin) gate (Fig. 1B, I). Lin + CD11b − cells, which include lymphocytes and plasmacytoid DCs, which can express CD4, CD8, and/or B220 7 , were gated forward (Fig. 1B, I). Plasmacytoid DCs were delineated from Lin + CD11b − cells as MHCII + CD11c + (Fig. 1E, L). From CD45 + Lin − cells, CD64 + cells were gated forward (Fig. 1C, J); microglia were CD45 dim Ly6C − and macrophages were CD45 high Ly6C mixed (Fig. 1D, K). From CD45 + Lin − cells, the CD64 − Ly6C − population was gated forward   Fig. 1F, M). Both CD11b − cDC-1 and CD11b + cDC-2 subsets were found (Fig. 1G, N). These data show that we can detect 3 DC population in the mouse eye. Please see our recent publication for our fluorescence minus one controls 11 . To identify the ocular sub-compartment of each DC subset, we dissected eyes into iris, choroid, and retina tissues. Each ocular tissue underwent multi-parameter flow cytometry with the gating strategy described in Fig. 1. At steady-state, we found similarly low numbers of plasmacytoid DCs in each tissue ( Fig. 2A). We found both cDC-1 and cDC-2 populations in iris and choroid, but could not detect cDCs in the retina (Fig. 2B,C). Since the laser-induced inflammatory lesion exists in the subretinal space, we analyzed DC heterogeneity in both retina and choroid tissues together on Day 3 after laser injury. Plasmacytoid DCs increased in both iris (4.8-fold, Figure 2. Each DC subtype was increased in the retina and choroid after laser injury. Number of plasmacytoid DCs (pDC, A), classical DC-1 (cDC-1), and classical DC-2 (cDC-2) identified in each ocular tissue at steady state. Number of pDC (D), cDC-1 (E), and cDC-2 (F) in either iris or retina and choroid in untreated (NC) or laser-treated mice. Number of pDC (G), c-DC1 (H), and c-DC2 (I) over time after laser injury (N = 9 per group). *p < 0.05; **p < 0.01; ***p < 0.001. www.nature.com/scientificreports/ p < 0.01) and choroid/retina (2.5-fold, p < 0.01) after laser injury (Fig. 2D). The number of cDC-1 cells was upregulated by 7.5-fold (p < 0.05) in iris and 9.6-fold in choroid/retina (p < 0.001) after laser injury (Fig. 2E). The cDC-2 population enlarged 5.5-fold (p < 0.01) in choroid/retina, but only a trend toward increased cDC-2 cells was found in the iris after laser treatment (Fig. 2F). These data demonstrate that each DC subset expands in the posterior segment after laser injury, while a similar but smaller increase is observed in the iris. We previously published that macrophage infiltration peaks on Day 3 after laser injury 10 . We performed multiparameter flow cytometry (identical to Fig. 1) on Day 3, Day 5, and Day 7 to investigate tissue infiltration of each DC subtype. This study was performed on whole eye samples (cornea, sclera, iris, ciliary body, lens, vitreous, retina, and choroid) to increase DC capture, maximize rigor and reproducibility, and minimize error created from uneven tissue dissections. We found that plasmacytoid DCs trended upward with a peak between Day 3-5 (Fig. 2G). Classical DC-1 cells increased 8.4-fold on Day 3 (p < 0.01 vs Day 0), 7.7-fold on Day 5 (p < 0.01 vs Day 0), and 3.8-fold on Day 7 (p < 0.05 vs Day 0, Fig. 2H). The cDC-2 population enlarged 5.7-fold on Day 3 (p < 0.01 vs Day 0), 5.6-fold on Day 5 (p < 0.01 vs Day 0), and 3.0-fold on Day 7 (p < 0.05 vs Day 0, Fig. 2I). These data demonstrate that the peak of DC infiltration is Day 3 similar to macrophages in the laser-induced CNV model.

Effect of Flt3 deficiency on CNV and DCs.
In order to investigate the function of DCs during laserinduced CNV, we first investigated the Flt3 −/− mouse. Fms-like tyrosine kinase 3 (Flt3) is a cell surface receptor that is necessary to maintain tissue resident DC populations via homeostatic DC proliferation 12 . Wildtype and Flt3 −/− male and female mice were treated with laser to break Bruch's membrane and induce CNV formation. On Day 14 after laser injury, we stained choroidal wholemounts with ICAM-2 to measure CNV area. We found no change in CNV area as a result of We next used multi-parameter flow cytometry to measure DC heterogeneity in the context of Flt3 deficiency. Since DC numbers per mouse were low and we found no significant difference in the response of each ocular sub-compartment to laser injury (Fig. 2), we performed our DC heterogeneity analysis in whole mouse eyes (cornea, sclera, iris, ciliary body, lens, vitreous, retina, and choroid). We chose Day 3 for multi-parameter flow cytometry studies, as it is the peak of both macrophage 10 and DC infiltration ( Fig. 2G-I) after laser injury. In females, macrophage numbers increased 12.8-fold (p < 0.01) in wildtype and 8.0-fold (p < 0.001) in Flt3 −/− mice after laser injury, with no significant difference between groups (Fig. 4A, E). Similarly, in males, macrophages www.nature.com/scientificreports/ expanded 5.9-7.3 fold after laser injury (p < 0.01 for both), with no significant differences between groups (Fig. 4J). Microglia numbers remained relatively unchanged regardless of sex, laser, or genotype ( Fig. 4A, F, K). In female mice, plasmacytoid DCs increased 5.1-fold (p < 0.001) in wildtype mice, but were not significantly increased in Flt3 −/− mice (Fig. 4B, G). Plasmacytoid DC numbers were unchanged by genotype or laser in male mice ( Fig. 4L). At steady-state, cDC-1 numbers decreased by 58% in female Flt3 −/− mice (p < 0.05, Fig. 4C, D, H), and were unchanged in male mice (Fig. 4M). After laser injury, the c-DC1 population was up-regulated by 5.6-9.7 fold (p < 0.001 for both, Fig. 4C, D, H) in female mice, and 2.6-3.5 fold (p < 0.05 for Flt3 −/− ) in male mice ( Fig. 4M) with no difference between genotypes . The steady-state cDC-2 population was significantly reduced in both sexes by 40-56% in Flt3 −/− mice (p < 0.05 for both, Fig. 4C, D, I, N). After laser injury, cDC-2 cells expanded by 5.1-7.8 fold in female mice (p < 0.001 for both, Fig. 4C, D, I) and 4.1-5.5 fold in male mice (p < 0.01 for both, Fig. 4N) with no significant differences between wildtype and Flt3 −/− mice. These data display that Flt3 −/− mice have reduced DCs at steady-state but are still capable of increasing their DC populations after laser injury.

Discussion
In this study, we show that DC heterogeneity exists in the eye, where we were able to detect classical DCs in the uvea, including the choroid and iris, but not in the retina (Fig. 2). Additionally, we found low numbers of plasmacytoid DCs in iris, choroid, and retina (Fig. 2). After laser injury, the number of each DC subtype increased in the posterior segment (Fig. 2). In order to determine the role of DCs during laser-induced CNV, we tested two models of DC deficiency. Flt3 −/− mice displayed a classical DC deficit at steady-state (Fig. 4), which resulted in no change in laser-induced CNV area (Fig. 3). Flt3l −/− mice demonstrated a severe DC absence at both steadystate and after laser injury (Fig. 6), which caused no significant reduction in laser-induced CNV area (Fig. 5). Therefore, DCs do not play a significant role in the laser-induced CNV model of nAMD.
Our results contradict a prior report where DCs were able to augment the laser-induced CNV model. In this study from 2008, bone marrow-derived cells were cultured for 9 days in the presence of granulocyte-macrophage colony-stimulating factor (Csf2) to mature MHCII + CD11c + cells 9 . These MHCII + CD11c + cells were injected systemically, and larger laser-induced CNV lesions were observed. Since this publication in 2008, several markers, including CD64, were found to reliably distinguish macrophages and DCs 14 . We have previously published, using CD64 to discriminate macrophages from DCs, that MHCII + CD11c + macrophage numbers are up-regulated during laser-induced CNV and express a pro-angiogenic transcriptome 10 . Therefore, the MHCII + CD11c + population investigated by Nakai et al. 9 likely included both macrophages and DCs, explaining the discrepancy in our results.
Flt3 −/− mice and intravitreal pharmacologic Flt3 blockade were previously shown to inhibit laser-induced CNV by ~ 40%, which we were not able to replicate 15 . The pharmacologic Flt3 inhibitor AC220 is specific at nanomolar levels, but the authors used 0.1-10 mg/ml (0.18-18 mM). At 6 orders of magnitude above specificity, AC220 has many off target effects including inhibition of platelet-derived growth factor receptors and colony stimulating factor 1 (Csf1) receptor 16 , which both can stimulate choroidal angiogenesis [17][18][19] . In addition, experiments in this prior publication were performed at 4-8 weeks of age, but the sex and genetic background of the Flt3 −/− mice were not reported. Our experiments were performed on both male (N = 9-13 per groups) and female (N = 10-11 per groups) mice, at 10-12 weeks of age, and the Flt3 −/− mice used in our study were on a C57BL/6 J background (Fig. 3). These discrepancies could explain the differences in results.
We were initially surprised that Flt3 −/− mice demonstrated DC deficits at steady-state only (Fig. 4), while Flt3l −/− mice showed severe DC deficiencies at both steady-state and in response to laser injury (Fig. 6). However, Flt3 −/− mice can exhibit an enhanced response to Csf1, leading to DC development 20 . Since Csf1 expression increases after laser injury 19 , Csf1-dependent signaling is a possible compensatory mechanism for DC maturation in Flt3 −/− mice, explaining why Flt3 −/− mice have increased DCs after laser injury.
A major limitation of our study is that it is very difficult to prove a negative hypothesis. We tested a single model of nAMD: the laser-induced CNV model. Additionally, we tested two models of DC deficiency: the  21 , it is more likely that the association between complement and AMD is not through DCs. In summary, we were able to detect plasmacytoid DCs, classical DC-1, and classical DC-2 subsets in the mouse choroid, and each subset was increased after laser injury. In the Flt3 −/− mouse model of steady-state DC deficiency, no change in laser-induced CNV area was detected. Finally, in the Flt3l −/− mouse model of severe DC deficiency at both steady-state and after laser injury, no significant difference in laser-induced CNV was found. Therefore, DCs play no significant role in the laser-induced CNV model. Mice were bred in-house and maintained in a pathogen-free barrier facility within Northwestern University's Center for Comparative Medicine. One complete litter from each breeding pair was genotyped to confirm the correct genotype and the absence of the RD8 allele (Crb1-). Genotyping services were performed by Transnetyx (Cordova, TN). All experiments were conducted in accordance with the ARRIVE guidelines, were approved by the Northwestern University Institutional Animal Care and Use Committee, and were performed in accordance with the relevant guidelines and regulations.
Laser-induced CNV. Male and female 10-12 week old mice underwent laser treatment as previously described 23 . Briefly, mice were anesthetized with ketamine/xylazine (Akorn, Lake Forest, IL) and received 1 mg/ kg subcutaneous injection of Meloxicam (Henry Schein Animal Health, Melville, NY). Eyes were anesthetized, dilated, and a cover slip was coupled to the cornea with Gonak (Akorn) for slit lamp biomicroscopy and laser. Four (immunofluorescence) or eight (flow cytometry, to increase inflammatory cell numbers) focal burns (75 μm, 100-120 mW, 100 ms) were administered in each eye using a 532 nm argon ophthalmic laser (IRIDEX, Mountain View, CA) via a slit lamp (Zeiss, Oberkochen, Germany).
Flow cytometry of whole eyes and ocular tissues. Experiments were performed as previously described 11 . Briefly, mice were sacrificed and eyes were enucleated and stored in cold HBSS. Animals did not undergo systemic perfusion because we previously showed no change in mononuclear phagocyte numbers at steady-state or after laser injury with or without systemic perfusion 11 . Eyes were dissected to remove optic nerve, extraocular muscles, orbital tissue, and conjunctiva. Remaining cornea, sclera, iris, ciliary body, vitreous, retina, and choroid were minced into small pieces. Tissues underwent further mechanical and chemical digestion, and were passed through a fine mesh filter to obtain a single cell suspension. Cell suspensions were stained with live/ dead and washed. Cell suspensions were blocked and then stained with fluorescently-conjugated antibodies ( Table 1). Both eyes were pooled from one mouse to analyze cells per mouse with counts beads as previously described 11 . For dissected iris, choroid, and retina, tissues were not minced, underwent chemical digestion for 1 h, and then were passed through a fine mesh filter. After the fine mesh filter, dissected and whole eye specimens were treated identically. Samples were run on a modified LSRII (BD Biosciences, San Jose, CA) and analyzed using FlowJo v10.
Statistical analysis. Flow cytometry comparisons between ocular tissues were made using One-Way ANOVA followed by Sidak's multiple comparisons test. Comparisons for CNV area were made using Student's unpaired t-test. Flow cytometry comparisons between wildtype and Flt3 −/− or Flt3l −/− mice, and over time, were made using the Brown-Forsythe and Welch ANOVA followed by Dunnett's T3 multiple comparison test due to unequal variances between unlasered and lasered mice.